The vast majority of all known two-component systems regulate gone expression in response to environmental stimuli with one notable exception being the chemotaxis system that regulates motility. Analysis of the Che3 chemosensory system in Myxococcus xanthus led to the surprising discovery, however, that this chemotaxis-like signal transduction system affects developmentally regulated gone expression without affecting motility or chemotaxis. Although the Che3 system comprises homologs to CheA, CheB, CheR, CheW, and two chemoreceptors (MCPs), the output of the system is unusual with respect to the prototype found in E. coli for the control of motility. The output of the Che3 system is not mediated by CheY but feeds through CrdA, a homolog of the NtrC family of sigma54-dependent transcriptional activators and thereby affects gene expression. Knowledge of the M. xanthus Che3 system will have important implications for our understanding of the broader fields of two-component signal transduction ranging from the control of prokaryotic development to biofilm formation and antibiotic resistance. The overall goal of this project is to fully characterize the signaling pathway comprising the M. xanthus Che3 system. To understand the mechanism of chemosensory regulation of gene expression and its role in development of M. xanthus, four specific aims are proposed: 1) characterize the phosphorelay component of the signaling pathway, 2) characterize the receptor signaling complex, 3) analyze the role of methylation-dependent adaptation, and 4) identify the output controlled by the Che3 system.